virus-like platform for rapid vaccine discovery

ABSTRACT

The invention is directed to virus-like particles (VLPs) of an RNA bacteriophage that (a) comprises a coat polypeptide of said phage modified by insertion of a heterologous peptide that is displayed on said VLP and (b) encapsidates said bacteriophage mRNA as well as populations of these VLPs, and their uses. The invention is further directed to VLPs that encapsidate heterologous substances, as well as populations of these VLPs and their uses.

PRIORITY CLAIM

This application claims priority from application Ser. No. 60/839,619,filed Aug. 23, 2006 and application Ser. No. 60/899,237, filed Feb. 2,2007, the contents of which are incorporated herein by reference.

GOVERNMENT INTEREST

This patent application was supported by grant NOS. RO1 GM04290 1 andR01 AI065240 from the National Institutes of Health. The Government hascertain rights in the invention.

FIELD OF THE INVENTION

The invention is directed to virus-like particles (VLPs) of an RNAbacteriophage that (a) comprises a coat polypeptide of said phagemodified by insertion of a heterologous peptide and said heterologouspeptide is displayed on said VLP and (b) encapsidates said bacteriophagemRNA. The invention is also directed to a population of these VLPs and acomposition comprising one or more of these VLPs and methods forobtaining these VLPs. Furthermore, the invention is directed to uses ofthese VLPs in identifying peptides of interest.

BACKGROUND OF THE INVENTION

The growth of recombinant DNA technology in recent years has led to theintroduction of vaccines in which an immunogenic protein has beenidentified, cloned and expressed in a suitable host to obtain sufficientquantities of protein to allow effective protective immunization in bothanimals and humans. Many of the most effective vaccines are based on thepotent ability of virion surfaces to elicit neutralizing antibodies.These include licensed killed or attenuated virus vaccines, such aspolio, influenza and rabies, which effectively induce protectiveantibody responses. More recently, subunit vaccines based uponself-assemblages of the structural proteins of human papillomavirus(HPV) and hepatitis B virus (HBV) have been approved by the Food andDrug Administration.

Phage display is one of several technologies that make possible thepresentation of large libraries of random amino acid sequences with thepurpose of selecting from them peptides with certain specific functions.The basic idea is to create recombinant bacteriophage genomes containinga library of randomized sequences genetically fused to one of thestructural proteins of the virion. When such recombinants aretransfected into bacteria each produces virus particles that display aparticular peptide on their surface and which package the samerecombinant genome that encodes that peptide, thus establishing thelinkage of genotype and phenotype essential to the method. Arbitraryfunctions (e.g. the binding of a receptor, immunogenicity) can beselected from such libraries by the use of biopanning and othertechniques. Because of constraints imposed by the need to transform andsubsequently cultivate bacteria, the practical upper limit on peptidelibrary complexity in phage display is said to be around 10¹⁰-10¹¹[Smothers et al., 2002, Science 298:621-622]. This requirement forpassage through E. coli is the result of the relatively complex makeupof the virions of the phages used for phage display, and the consequentnecessity that their components be synthesized and assembled in vivo.For example, display of certain peptides is restricted when filamentousphage is used, or not possible, since the fused peptide has to besecreted through the E. coli membranes as part of the phage assemblyapparatus.

SUMMARY OF THE INVENTION

The invention is directed to a population or library of virus-likeparticles (VLPs), wherein each particle (a) is a VLP of an RNAbacteriophage, (b) comprises a coat polypeptide of said bacteriophagemodified by insertion of a heterologous peptide wherein saidheterologous peptide is displayed on said bacteriophage and (c)encapsidates said bacteriophage mRNA. In a particular embodiment, theVLPs are VLPs of an MS2 RNA bacteriophage and/or the coat polypeptide isa single chain dimer containing an upstream or downstream subunit whereoptionally the heterologous peptide is inserted either in the upstreamor preferably downstream subunit or alternatively, the N-terminus orC-terminus of the coat polypeptide. In a particular embodiment, theheterologous peptide is at least four amino acid sequences in length. Inyet another particular embodiment, at least 90 copies of saidheterologous peptide is displayed on said VLP; in yet a furtherembodiment, between 1-180 copies of said heterologous peptide isdisplayed on said VLP.

The population or library of VLP particles of the present invention maybe obtained by providing a plurality of transcription units comprising abacterial or bacteriophage promoter, a coding sequence of an RNAbacteriophage single chain coat polypeptide dimer with a site forinsertion of a heterologous peptide in the downstream or upstreamsubunit of the dimer and bacterial or bacteriophage terminator; (b)treating said transcription units of (a) with a restriction enzyme; (c)inserting coding sequences for heterologous peptides into saidtranscription units to obtain a population of transcription units; (d)expressing said transcription units of (c) and (e) isolating saidlibrary. In a specific embodiment, the invention comprises: (a)providing a transcription unit comprising a bacteriophage promoter, acoding sequence for a modified RNA bacteriophage coat polypeptide,wherein said modification is a heterologous peptide sequence, optionallyat least 4 amino acid sequences in length, and optionally abacteriophage terminator; (b) expressing said transcription unit in acoupled transcription/translation system from a nucleic acid templateoptionally in a compartmentalized water/oil emulsion and (c) recoveringsaid population from said transcription/translation system.

The invention is further directed to the isolated transcription unitsmentioned above. In a specific embodiment, the transcription unitcomprises a bacterial or bacteriophage promoter, a coding sequence of anRNA bacteriophage single chain coat polypeptide with a site forinsertion of a heterologous peptide in said coding sequence andoptionally bacteriophage terminator. In a more specific embodiment, thecoat polypeptide is a single chain coat polypeptide dimer with anupstream subunit and downstream subunit with a site for insertion of aheterologous peptide in either the upstream or downstream subunit of thedimer. In a particular embodiment, the heterologous peptide is insertedin the downstream subunit. In yet another embodiment, the transcriptionunit is free of translational operator sequence (also referred to hereinas coat recognition site, packaging signal, RNA binding site,translational operator signal).

Additionally, the population of the present invention may be used toidentifying a peptide having a property of interest. This methodcomprises: (a) providing the population or library of the presentinvention and (b) assaying heterologous peptides expressed on the VLPsin the population of the present invention for the property of interestto identify the peptide of interest. The property of interest may beimmunogenicity (e.g., ability to act as an eptiope or mimitope),pharmacological effectiveness, ability to bind to filamentous phage,ability to bind to a cell surface receptor.

In a related aspect, the invention is directed to a method for isolatingan immunogenic protein comprising (a) identifying said immunogenicpeptide from a population of VLPs according to the method of the presentinvention; (b) amplifying said identified immunogenic peptide and (c)isolating said immunogenic peptide. In a particular embodiment, theimmunogenic peptide is an immunogenic fragment of a self-antigen.Alternatively, the immunogenic peptide, the immunogenic peptide is afragment of an immunogenic HIV peptide.

The invention is also directed to an isolated VLP of an RNAbacteriophage which comprises a single-chain dimer of coat polypeptideof said phage modified by insertion of a heterologous peptide,optionally at least 4 amino acids in length, wherein said heterologouspeptide is displayed on said VLP, wherein said heterologous peptide isselected from the group consisting of an HIV peptide, a self antigen, areceptor and a ligand which binds to a cell surface receptor, a peptidewith affinity for either end of a filamentous phage particle specificpeptide, metal binding peptide, a peptide with affinity for saidbacteriophage surface and/or promotes self-assembly. In a relatedaspect, the invention is directed to a composition comprising one ormore of said isolated VLPs. In one particular embodiment, the VLPcomprises a modified coat polypeptide comprising a pharmaceuticallyeffective heterologous polypeptide coupled to a ligand for binding to acell receptor. In another related aspect, the VLPs coupled to adetectable label (e.g., metal chelator, biotin). In another relatedaspect, the invention is directed to a composition comprising one ormore of the VLPs of the present invention.

In a more specific embodiment, the invention is directed to animmunogenic composition comprising one or more VLPs of a MS2 RNAbacteriophage and comprises a single chain dimer of the coat polypeptideof said phage, said coat polypeptide comprising an upstream anddownstream subunit, wherein said upstream or downstream subunit ismodified by insertion of an immunogenic heterologous peptide in eitherthe upstream or downstream subunit of said dimer. The immunogeniccomposition may be a vaccine.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows a schematic representation of the 3569-nucleotide genomeof RNA bacteriophage MS2. FIG. 1B shows the sequence of thetranslational operator (SEQ ID NO:1).

FIG. 2 shows a schematic representation of one design for atranscription/translation template capable of producingRNA-bacteriophage-like particles displaying foreign peptides on theirsurface while encapsidating the RNA that encodes the coatpolypeptide-peptide fusion.

FIG. 3 shows principal features of a VLP transcription unit.

FIG. 4 shows a method for the construction of a library of 10-amino acidinsertions in the downstream-most AB-loop of the single-chain dimer. Theprocedure starts with plasmids pET2MCTK3 (see FIG. 16) and pCT119c15N(see FIG. 17) and produces a vector fragment by restriction enzymedigestion, and an insert fragment by PCR. One primer (P3 having thesequence 5′-GTTGTAAAACGACGGCCAGT-3′ depicted in SEQ ID NO:13 annealsdownstream of a unique BamHl site and the other (PN30) having thesequence depicted in SEQ ID NO:14,5′-CGCGGTACCNNSNNSNNSNNSNNSNNSNNSNNSNNSNNSGGAACTGGCGACGTGA CTGTC-3′,where S=G or C and N=A,C,G,T primes immediately downstream of theplasmid's Kpn I site (eliminating it) and introduces a random30-nucleotide sequence and a new Kpn I site at the 5′-end end of the newfragment. These fragments are joined by ligation and introduced into E.coli by electroporation to produce a complex library of random peptidesequences.

FIG. 5 shows a strategy for rapid vaccine discovery.

FIG. 6 shows the SDS-polyacrylamide gel electrophoresis of the productsof in vitro transcription/translation.

FIG. 7 shows an agarose gel electrophoresis to detect capsids producedby transcription/translation.

FIG. 8. (A) Structure of the MS2 coat polypeptide dimer seen edge-on.The two polypeptide chains are colored red and blue and the AB-loops areemphasized by showing amino acids 13, 14 and 15 in a space-fillingrepresentation. (B) The MS2 VLP with coat polypeptide subunits in greenand the AB-loops in red space-fill to illustrate their arrangement andexposure on the VLP surface. (C) Structure of the MS2 coat polypeptidedimer as it would be viewed from outside the capsid. Note the proximityof the N- and C-termini of the dimer's two polypeptide chains.

FIG. 9 shows the single-chain dimer was constructed by genetic fusion ofthe coat polypeptide-s two identical polypeptide chains.

FIG. 10(A) shows arrangements of the coat polypeptide reading frames ofthe plasmids used in this study. All express coat polypeptide from thelac promoter. pMCTK is similar to the previously described pCT119(Chackerian et al., 2001, J Clin Invest 108(3):415-23 wrong reference.Replace with Peabody 1990, J. Biol, Chem. 265: 5684-5689), but hassilent mutations in codons 14 and 15 that introduce the Kpn I site(indicated by arrows). p2MCTK3 expresses a single-chain dimer version ofthe protein with the Kpn I site in the C-terminal half of thesingle-chain dimer. Black boxes represent the ECL2 or V3 peptideinsertions in the various plasmid derivatives. FIG. 10(B) Amino acidsequences (single-letter code) of the ECL2 and V3 peptides (top lines)and of the annealed oligonucleotides that encode them (SEQ ID NOS: 4-9).

FIG. 11(A) Western blot analysis of the proteins produced by the variousconstructs described in the text and illustrated schematically here. Ineach case, a cell lysate was produced by sonication and then segregatedinto soluble (S) and insoluble (or pellet, P) fractions bycentrifugation. Coat proteins were visualized using rabbit anti-MS2serum and an alkaline phosphatase-labeled second antibody. FIG. 11(B)show elution of coat proteins from Sepharose CL-4B. Cell extracts wereapplied to the column and the coat protein content of individualfractions was determined by SDS-polyacrylamide gel electrophoresis.Proteins were visualized in the gel both by Coomassie Blue staining andby Western Blot, and the quantity of coat in each fraction wasdetermined by densitometry. Authentic MS2 virus co-elutes with the VLPsproduced by the recombinants. FIG. 11(C) shows agarose gelelectrophoresis of purified bacteriophage MS2 and the VLPs produced bythe single-chain dimer construct and by the ECL2 and V3 recombinantVLPs. Protein was stained with Coomassie Blue R250 (left). Because theVLPs contain RNA they can also be visualized with ethidium bromide (atright).

FIG. 12(A) shows an anti-V3 mAb binds to V3-VLPs, but not ECL2-VLPs orwild-type MS2 VLPs. Dilutions of MAbIIIB-V3-13 were reacted with 500ng/well of V3-VLPs (ν), wild-type MS2 VLPs (O), or ECL2-VLPs (λ).Binding was detected using a horseradish peroxidase-labeled goatanti-mouse IgG secondary followed by development with ABTS. Reactivitywas measured by optical density at 405 nm (OD₄₀₅). FIG. 12 (B) IgGantibody responses in C57Bl/6 mice immunized with wild-type MS2 VLPs,V3-VLPs, or ECL2-VLPs. End-point dilution ELISA titers against a peptiderepresenting HIV gp120 V3 (left panel), or a peptide representing theCCR5 ECL2 undecapeptidyl arch (UPA) (right panel) in serum from miceimmunized three times with each VLP type. VLPs were administered eitherin the presence of complete Freund's adjuvant (CFA) or without adjuvant(NA). Results are from sera obtained 7 days after the third vaccination.Each data point represents the antibody titer from an individual mouse.Lines represent the geometric mean titer for each group. FIG. 12 (C)Neutralization of HIV-1_(LAI) infection of the MAGI-CCR5 indicator cellline using sera from mice immunized with V3-VLPs. Approximately 100infectious virus particles were incubated with dilutions of pooled serafrom mice immunized with wild-type MS2 VLPs, pooled sera from miceimmunized with V3-VLPs, or the HIV neutralizing mAbs V3-13 or b12 (apotent neutralizing monoclonal antibody that recognizes an epitopeoutside of the V3 domain) for 1 h and then added to target cells. Twodays after infection, infected cells were scored by counting the numberof blue cells in each well. Inhibition of HIV infection was determinedby comparing the number of blue (infected) nuclei in the presence ofantibody versus the number of blue nuclei in the absence of antibody.Data represents the average of two different experiments; error barsshow standard error of the mean. FIG. 12(D) Flow cytometric analysis ofantibody binding to transiently transfected 293T cells. Cells weremock-transfected (shaded histogram) or transfected with pc.Rh-CCR5(thick solid line) and then incubated with (upper left) a PE-labeledanti-CCR5 mAb (3A9), (upper right) secondary antibody alone, (lowerleft) sera from a mouse immunized with wild-type MS2 VLPs, or (lowerright) sera from a mouse immunized with ECL2-VLPs.

FIG. 13 shows a hydrophobicity plot by the method of Kyte and Doolittleof a hypothetical protein produced by arbitrarily grouping all therandom peptide sequences that resulted in a repressor-competent coatprotein (residues 1-482) and those that interfered with coat function(residues 483-662). Note the sharp transition to higher hydrophobicityat about amino acid 483.

FIG. 14 shows electrophoresis on agarose gel of VLPs found in crudelysates of cells containing various recombinant plasmids, each of whichproduces a coat protein with a different random 6-, 8- or 10-amino acidinsertion. The first two lanes of each gel are controls: pUCter3produces no coat protein, while p2MCTK3 is the single-chain dimerconstruct without a peptide insertion. Ethidium bromide-stained gels(upper half of each set) and blots probed with anti-MS2 serum (lowerhalf of each set) are shown.

FIG. 15 shows electrophoresis (left panel) and Northern Blot analysis(right panel) of VLP-encapsdated RNAs. CT VLP and 2CT VLP refer to theRNAs extracted from conventional and single-chain dimer VLPsrespectively. CT in vitro and 2CT in vitro refer to the products oftranscription in vitro of the same plasmids that produced the VLPs. Thein vitro transcription product of a similar plasmid containing HCV coresequences (core in vitro) was also run for comparison. MS2 and Qβ RNAswere extracted from the purified phages.

FIG. 16 shows a schematic diagram of pET2MK3 depicted in SEQ ID NO:18.pET2MK3 has the coat sequence of p2MK3 under T7 promoter control. It hasa T7 transcription terminator, but no packaging signal.

FIG. 17 shows a schematic diagram of pCT119-d15N depicted in SEQ IDNO:19. This plasmid has the coat sequence under lac promoter control. Itserves as a convenient source of template for PCR reactions to generaterandom sequence peptide libraries.

FIG. 18 shows a schematic diagram of pMCTK2 depicted in SEQ ID NO:20.This plasmid is a pUC119 derivative, contains an E. coli lac promoterwhich drives transcription of a coat sequence with a unique KpnI site incodons 14 and 15 and has the translational operator just downstream ofcoat gene.

FIG. 19 shows a schematic diagram of p2MCTK3. This plasmid is a pUC119derivative, contains an E. coli lac promoter which drives transcriptionof a single-chain dimer coat sequence with a unique KpnI site in codons14 and 15 of the downstream copy of the coat sequence and has thetranslational operator (packaging signal) just downstream of coat gene.

FIG. 20 shows a schematic diagram of pETCT depicted in SEQ ID NO:21.pETCT contains a T7 promoter which drives transcription of the wild-typecoat gene and a T7 transcription terminator downstream of coat.

FIG. 21 shows a schematic diagram of pET2CT. pETCT contains a T7promoter which drives transcription of the single-chain dimmer sequenceand a T7 transcription terminator downstream of coat.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, 2001, “MolecularCloning: A Laboratory Manual”; Ausubel, ed., 1994, “Current Protocols inMolecular Biology” Volumes I-III; Celis, ed., 1994, “Cell Biology: ALaboratory Handbook” Volumes I-III; Coligan, ed., 1994, “CurrentProtocols in Immunology” Volumes I-III; Gait ed., 1984, “OligonucleotideSynthesis”; Hames & Higgins eds., 1985, “Nucleic Acid Hybridization”;Hames & Higgins, eds., 1984, “Transcription And Translation”; Freshney,ed., 1986, “Animal Cell Culture”; IRL Press, 1986, “Immobilized CellsAnd Enzymes”; Perbal, 1984, “A Practical Guide To Molecular Cloning.”

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and” and “the” include plural references unless thecontext clearly dictates otherwise.

Furthermore, the following terms shall have the definitions set outbelow.

As used herein, the term “polynucleotide” refers to a polymeric form ofnucleotides of any length, either ribonucleotides or deoxynucleotides,and includes both double- and single-stranded DNA and RNA. Apolynucleotide may include nucleotide sequences having differentfunctions, such as coding regions, and non-coding regions such asregulatory sequences (e.g., promoters or transcriptional terminators). Apolynucleotide can be obtained directly from a natural source, or can beprepared with the aid of recombinant, enzymatic, or chemical techniques.A polynucleotide can be linear or circular in topology. A polynucleotidecan be, for example, a portion of a vector, such as an expression orcloning vector, or a fragment.

As used herein, the term “polypeptide” refers broadly to a polymer oftwo or more amino acids joined together by peptide bonds. The term“polypeptide” also includes molecules which contain more than onepolypeptide joined by a disulfide bond, or complexes of polypeptidesthat are joined together, covalently or noncovalently, as multimers(e.g., dimers, tetramers). Thus, the terms peptide, oligopeptide, andprotein are all included within the definition of polypeptide and theseterms are used interchangeably. It should be understood that these termsdo not connote a specific length of a polymer of amino acids, nor arethey intended to imply or distinguish whether the polypeptide isproduced using recombinant techniques, chemical or enzymatic synthesis,or is naturally occurring.

The amino acid residues described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfunctional is retained by the polypeptide. NH₂ refers to the free aminogroup present at the amino terminus of a polypeptide. COOH refers to thefree carboxy group present at the carboxy terminus of a polypeptide.

The term “coding sequence” is defined herein as a portion of a nucleicacid sequence which directly specifies the amino acid sequence of itsprotein product. The boundaries of the coding sequence are generallydetermined by a ribosome binding site (prokaryotes) or by the ATG startcodon (eukaryotes) located just upstream of the open reading frame atthe 5′-end of the mRNA and a transcription terminator sequence locatedjust downstream of the open reading frame at the 3′-end of the mRNA. Acoding sequence can include, but is not limited to, DNA, cDNA, andrecombinant nucleic acid sequences.

A “heterologous” region of a recombinant cell is an identifiable segmentof nucleic acid within a larger nucleic acid molecule that is not foundin association with the larger molecule in nature.

An “origin of replication” refers to those DNA sequences thatparticipate in DNA synthesis.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation, as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase. Eukaryotic promoters will often, but not always, contain“TATA” boxes and “CAT” boxes. Prokaryotic promoters containShine-Dalgarno sequences in addition to the −10 and −35 consensussequences.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence. Transcriptional andtranslational control sequences are DNA regulatory sequences, such aspromoters, enhancers, polyadenylation signals, terminators, and thelike, that provide for the expression of a coding sequence in a hostcell.

A “signal sequence” can be included before the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide, thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA.

It should be appreciated that also within the scope of the presentinvention are nucleic acid sequences encoding the polypeptide(s) of thepresent invention, which code for a polypeptide having the same aminoacid sequence as the sequences disclosed herein, but which aredegenerate to the nucleic acids disclosed herein. By “degenerate to” ismeant that a different three-letter codon is used to specify aparticular amino acid.

As used herein, “epitope” refers to an antigenic determinant of apolypeptide. An epitope could comprise 3 amino acids in a spatialconformation which is unique to the epitope. Generally an epitopeconsists of at least 5 such amino acids, and more usually, consists ofat least 8-10 such amino acids. Methods of determining the spatialconformation of amino acids are known in the art, and include, forexample, x-ray crystallography and 2-dimensional nuclear magneticresonance.

As used herein, a “mimitope”, is a peptide that mimics an authenticantigenic epitope.

As used herein, the term “coat protein(s)” refers to the protein(s) of abacteriophage or a RNA-phage capable of being incorporated within thecapsid assembly of the bacteriophage or the RNA-phage.

As used herein, a “coat polypeptide” as defined herein is a polypeptidefragment of the coat protein that possesses coat protein function andadditionally encompasses the full length coat protein as well orsingle-chain variants thereof.

As used herein, the term “immune response” refers to a humoral immuneresponse and/or cellular immune response leading to the activation orproliferation of B- and/or T-lymphocytes and/or and antigen presentingcells. In some instances, however, the immune responses may be of lowintensity and become detectable only when using at least one substancein accordance with the invention. “Immunogenic” refers to an agent usedto stimulate the immune system of a living organism, so that one or morefunctions of the immune system are increased and directed towards theimmunogenic agent. An “immunogenic polypeptide” is a polypeptide thatelicits a cellular and/or humoral immune response, whether alone orlinked to a carrier in the presence or absence of an adjuvant.Preferably, antigen presenting cell may be activated.

As used herein, the term “self antigen” refers to proteins encoded bythe host's DNA and products generated by proteins or RNA encoded by thehost's DNA are defined as self. In addition, proteins that result from acombination of two or several self-molecules or that represent afraction of a self-molecule and proteins that have a high homology twoself-molecules as defined above (>95%, preferably >97%, morepreferably >99%) may also be considered self.

As used herein, the term “vaccine” refers to a formulation whichcontains the composition of the present invention and which is in a formthat is capable of being administered to an animal.

As used herein, the term “virus-like particle of a bacteriophage” refersto a virus-like particle (VLP) resembling the structure of abacteriophage, being non replicative and noninfectious, and lacking atleast the gene or genes encoding for the replication machinery of thebacteriophage, and typically also lacking the gene or genes encoding theprotein or proteins responsible for viral attachment to or entry intothe host.

This definition should, however, also encompass virus-like particles ofbacteriophages, in which the aforementioned gene or genes are stillpresent but inactive, and, therefore, also leading to non-replicativeand noninfectious virus-like particles of a bacteriophage.

VLP of RNA bacteriophage coat protein: The capsid structure formed fromthe self-assembly of between 1-180 subunits of RNA bacteriophage coatprotein and optionally containing host RNA is referred to as a “VLP ofRNA bacteriophage coat protein”.

A nucleic acid molecule is “operatively linked” to an expression controlsequence when the expression control sequence controls and regulates thetranscription and translation of nucleic acid sequence. The term“operatively linked” includes having an appropriate start signal (e.g.,ATG) in front of the nucleic acid sequence to be expressed andmaintaining the correct reading frame to permit expression of thenucleic acid sequence under the control of the expression controlsequence and production of the desired product encoded by the nucleicacid sequence. If a gene that one desires to insert into a recombinantDNA molecule does not contain an appropriate start signal, such a startsignal can be inserted in front of the gene.

The term “stringent hybridization conditions” are known to those skilledin the art and can be found in Current Protocols in Molecular Biology,John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limitingexample of stringent hybridization conditions is hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 50° C., preferably at 55° C., andmore preferably at 60° C. or 65° C.

Production of Virus-Like Particles

The present invention is directed to virus-like phage particles as wellas methods for producing these particles in vitro. The resulting phagecan be used to conduct phage display in vitro The invention makes itpossible to increase laboratory complexity and reduce the time neededfor iterative selection. The methods typically include producing virionsin vitro and recovering the virions. As used herein, producing virions“in vitro” refers to producing virions outside of a cell, for instance,in a cell-free system, while producing virions “in vivo” refers toproducing virions inside a cell, for instance, an Eschericia coli orPseudomonas aeruginosa cell.

Bacteriophages

The system envisioned here is based on the properties of single-strandRNA bacteriophages [RNA Bacteriophages, in The Bacteriophages. Calendar,RL, ed. Oxford University Press. 2005]. The known viruses of this groupattack bacteria as diverse as E coli, Pseudomonas and Acinetobacter.Each possesses a highly similar genome organization, replicationstrategy, and virion structure. These include but are not limited toMS2, Qβ, R17, SP, PP7, GA, M11, MX1 and f2.

For purposes of illustration, the genome of a particularlywell-characterized member of the group, called MS2, is shown in FIG. 1A.It is a single strand of (+)-sense RNA 3569 nucleotides long, encodingonly four proteins, two of which are structural components of thevirion. The viral particle is comprised of an icosahedral capsid made of180 copies of coat protein and one molecule of maturase protein togetherwith one molecule of the RNA genome. Coat protein is also a specific RNAbinding protein. Assembly may possibly be initiated when coat proteinassociates with its specific recognition target an RNA hairpin near the5′-end of the replicase cistron (FIG. 1B) as shown in SEQ ID NO:1. Thevirus particle is then liberated into the medium when the cell burstsunder the influence of the viral lysis protein. The formation of aninfectious virus requires at least three components, namely coatprotein, maturase and viral genome RNA, but experiments show that theinformation required for assembly of the icosahedral capsid shell iscontained entirely within coat protein itself. For example, purifiedcoat protein can form capsids in vitro in a process stimulated by thepresence of RNA [Beckett et al., 1988, J. Mol. Biol 204: 939-47].Moreover, coat protein expressed in cells from a plasmid assembles intoa virus-like particle in vivo [Peabody, D. S., 1990, J Biol Chem 265:5684-5689].

Coat Polypeptide

The coat polypeptide encoded by the coding region is typically at least120, preferably, at least 125 amino acids in length, and no greater than135 amino acids in length, preferably, no greater than 130 amino acidsin length. It is expected that a coat polypeptide from essentially anysingle-stranded RNA bacteriophage can be used. Examples of coatpolypeptides include but are not limited to the MS2 coat polypeptide(see, for example SEQ ID NO:2), R17 coat polypeptide (see, for example,Genbank Accession No P03612), PRR1 coat polypeptide (see, for example,Genbank Accession No. ABH03627), fr phage coat polypeptide (see, forexample, Genbank Accession No. NP_(—)039624), GA coat polypeptide (see,for example, Genbank Accession No. P07234), Qβ coat polypeptide (see,for example, Genbank Accession No. P03615), SP coat polypeptide (see,for example, Genbank Accession No P09673), and PP7 coat polypeptide(see, for example, Genbank Accession No PO363 0).

The coat polypeptides useful in the present invention also include thosehaving similarity with one or more of the coat polypeptide sequencesdisclosed above. The similarity is referred to as structural similarityand is generally determined by aligning the residues of the two aminoacid sequences (i.e., a candidate amino acid sequence and the amino acidsequence, for instance, of SEQ ID NO: 2) to optimize the number ofidentical amino acids along the lengths of their sequences; gaps ineither or both sequences are permitted in making the alignment in orderto optimize the number of identical amino acids, although the aminoacids in each sequence must nonetheless remain in their proper order. Acandidate amino acid sequence is the amino acid sequence being comparedto an amino acid sequence present in SEQ ID NO: 2. A candidate aminoacid sequence can be isolated from a single stranded RNA virus, or canbe produced using recombinant techniques, or chemically or enzymaticallysynthesized. Preferably, two amino acid sequences are compared using theBESTFIT algorithm in the GCG package (version 10.2, Madison Wis.), orthe Blastp program of the BLAST 2 search algorithm, as described byTatusova, et al. (FEMS Microbial Lett 1999, 174:247-250), and availableat http://www.ncbi.nlm.nih.gov/blast/b12seq/b12.html. Preferably, thedefault values for all BLAST 2 search parameters are used, includingmatrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gapxdropoff=50, expect=10, wordsize=3, and optionally, filter on. In thecomparison of two amino acid sequences using the BLAST search algorithm,structural similarity is referred to as “identities.” Preferably, a coatpolypeptide also includes polypeptides with an amino acid sequencehaving at least 80% amino acid identity, at least 85% amino acididentity, at least 90% amino acid identity, or at least 95% amino acididentity to one or more of the amino acid sequences disclosed above.Preferably, a coat polypeptide is active. Whether a coat polypeptide isactive can be determined by evaluating the ability of the polypeptide toform a capsid and package a single stranded RNA molecule. Such anevaluation can be done using an in vivo or in vitro system, and suchmethods are known in the art and routine.

Heterologous peptide sequences inserted into the coat polypeptide orpolypeptide may be a peptide sequence that includes Xaa_(n), wherein nis at least 4, at least 6, or at least 8 and no greater than 20, nogreater than 18, or no greater than 16, and each Xaa is independently arandom amino acid. Alternatively, the peptide fragment may possess aknown functionality (e.g., antigenicity, immunogenicity). Theheterologous sequence may be present at the amino-terminal end of a coatpolypeptide, at the carboxy-terminal end of a coat polypeptide, orpresent elsewhere within the coat polypeptide. Preferably, theheterologous sequence is present at a location in the coat polypeptidesuch that the insert sequence is expressed on the outer surface of thecapsid. In a particular embodiment, the peptide sequence may be insertedinto the A-B loop regions the above-mentioned coat polypeptides.Examples of such locations include, for instance, insertion of theinsert sequence into a coat polypeptide immediately following aminoacids 11-17, or amino acids 113-117 of the coat polypeptide. In a mostparticular embodiment, the heterologous peptide is inserted at a sitecorresponding to

(a) amino acids 11-17 or particularly 13-17 of MS-2, R17 and fr coatpolypeptides;

(b) amino acids 10-16 of GA coat polypeptide

(c) amino acids 10-17 of QB and SP coat polypeptides;

(d) amino acids 8-11 of PP7 coat polypeptides and

(e) amino acids 9-17 of PRR1 coat polypeptides.

Alternatively, the heterologous peptide may be inserted at theN-terminus or C-terminus of the coat polypeptide.

In order to determine a corresponding position in a structurally similarcoat polypeptide, the amino acid sequence of this structurally similarcoat polypeptide is aligned with the sequence of the named coatpolypeptide as specified above in the section entitled Amino AcidHomology. For example, the corresponding position of a coat polypeptidestructurally similar to MS-2 coat polypeptide is aligned with SEQ IDNO:2. From this alignment, the position in the other coat polypeptidewhich corresponds to a given position of SEQ ID NO:1 can be determined.

In a particular embodiment, the coat polypeptide is a single-chain dimercontaining an upstream and downstream subunit Each subunit contains afunctional coat polypeptide sequence. The heterologous peptide may beinserted ton the upstream and/or downstream subunit at the sitesmentioned herein above, e.g., A-B loop region of downstream subunit. Ina particular embodiment, the coat polypeptide is a single chain dimer ofan MS2 coat polypeptide which may have a sequence depicted in SEQ IDNO:12.

Preparation of Transcription Unit

The transcription unit of the present invention comprises an expressionregulatory region, (e.g., a promoter), a sequence encoding a coatpolypeptide and transcription terminator. The RNA polynucleotide mayoptionally include a coat recognition site (also referred to a“packaging signal”, “translational operator sequence”, “coat recognitionsite”). A most particular embodiment is shown in FIG. 2. Alternatively,the transcription unit may be free of the translational operatorsequence. The promoter, coding region, transcription terminator, and,when present, the coat recognition site, are generally operably linked.Operably linked” refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended manner. A regulatory sequence is “operably linked” to a codingregion when it is joined in such a way that expression of the codingregion is achieved under conditions compatible with the regulatorysequence. The coat recognition site, when present, may be at anylocation within the RNA polynucleotide provided it functions in theintended manner.

The invention is not limited by the use of any particular promoter, anda wide variety of promoters are known. The promoter used in theinvention can be a constitutive or an inducible promoter. Preferredpromoters are able to drive high levels of RNA encoded by me codingregion encoding the coat polypeptide Examples of such promoters areknown in the art and include, for instance, T7, T3, and SP6 promoters.

The nucleotide sequences of the coding regions encoding coatpolypeptides described herein are readily determined. An example of theclass of nucleotide sequences encoding one of the coat polypeptidesdescribed herein is nucleotides 4080-4470 of SEQ ID NO:3. These classesof nucleotide sequences are large but finite, and the nucleotidesequence of each member of the class can be readily determined by oneskilled in the art by reference to the standard genetic code.

Furthermore, the coding sequence of an RNA bacteriophage single chaincoat polypeptide comprises a site for insertion of a heterologouspeptide as well as a coding sequence for the heterologous peptideitself. In a particular embodiment, the site for insertion of theheterologous peptide is a restriction enzyme site.

In a particular embodiment, the coding region encodes a single-chaindimer of the coat polypeptide. In a most particular embodiment, thecoding region encodes a modified single chain coat polypeptide dimer,where the modification comprises an insertion of a coding sequence atleast four amino acids at the insertion site. A schematic diagram of aparticular embodiment of such a transcription unit is shown in FIG. 3.The transcription unit may contain a bacterial promoter, such as a lacpromoter or it ma contain a bacteriophage promoter, such as a T7promoter and optionally a T7 transcription terminator.

In addition to containing a promoter and a coding region encoding afusion polypeptide, the RNA polynucleotide typically includes atranscription terminator, and optionally, a coat recognition site. Acoat recognition site is a nucleotide sequence that forms a hairpin whenpresent as RNA. This is also referred to in the art as a translationaloperator, a packaging signal, and an RNA binding site. Without intendingto be limiting, this structure is believed to act as the binding siterecognized by the translational repressor (e.g., the coat polypeptide),and initiate RNA packaging. The nucleotide sequences of coat recognitionsites are known in the art and include, for instance, nucleotides in SEQID NO:1 (see FIG. 1B). Other coat recognition sequences have beencharacterized in the single stranded RNA bacteriophages R17, GA, Qβ, SP,and PP7, and are readily available to the skilled person. Essentiallyany transcriptional terminator can be used in the RNA polynucleotide,provided it functions with the promoter. Transcriptional terminators areknown to the skilled person, readily available, and routinely used.

Synthesis

As will be described in further detail below, the VLPs of the presentinvention may be synthesized in vitro in a coupled cell-freetranscription/translation system. Alternatively VLPs could be producedin vivo by introducing transcription units into bacteria, especially iftranscription units contain a bacterial promoter.

VLP Populations

As noted above, the invention is directed to VLP populations orlibraries. The terms “population” and “libraries” in the instantspecification are used interchangeably and are thus deemed to besynonymous. In one particular embodiment, the library may be a randomlibrary; in another embodiment, the library is an antigen fragmentlibrary, a library of fragments derived from an antigenic polypeptide.

Random Libraries (Populations)

Oligonucleotides encoding peptides containing may be prepared. In oneparticular embodiment, In a particular embodiment, the triplets encodinga particular amino acid has the composition NNS where N is A, G, C or Tand S is G or T or alternatively NNY where N is A, G, C, or T and Y is Cor T. In order to minimize the presence of stop codons, peptidelibraries can be constructed using oligonucleotides synthesized fromcustom trinucleotide phosphoramidite mixtures (available from GlenResearch, Inc.) designed to more accurately reflect natural amino acidcompositions and completely lacking stop codons.

FIG. 4 shows a simple scheme for producing a library of 10-amino acidinsertions in the AB-loop of the C-terminal copy of a single-chaindimer. As shown in FIG. 4, a KpnI site engineered into codons 14 and 15of the downstream coat sequence of the single-chain dimer In anotherembodiment, alternate plasmids may be constructed which take advantageof a naturally occurring SalI site at codons 11 and 12. The SalI sitesmay be removed from all other locations in the plasmid, including theone normally present in the upstream half of the single-chain dimer, sonow a unique SalI site is found in the downstream half. In such aninstance, the PN30 primer may have the sequence5′-CCCCGTCGACAATGGCNNSNNSNNSNNSNNSNNSNNSNNSGGAACTGGCGACGTG ACTGTC-3′(SEQ ID NO:15) and would result in the insertion of random 8-amino acidsequences between amino acids 13 and 14. A primer of sequence5′-CCCCGTCGACAATGGCNNSNNSNNSNNSNNSNNSNNSNNGGCGACGTGACTGTCG CCCCA-3′ (SEQID NO:16) inserts random 8-amino acid peptides between amino acids 13and 16, and 5′-CCCCGTCGACAATNNSNNSNNSNNSNNSNNSNNSNNSGACGTGACTGTCGCCCCAAGC-3′ (SEQ ID NO:17) puts them between amino acids 12 and 17.

Antigenic Libraries

An alternative strategy takes advantage of the existence of a clonedantigen gene or pathogen genome to create random antigen fragmentlibraries. The idea is to randomly fragment the gene (e.g. with DNasel)to an appropriate average size (e.g. −30 bp), and to blunt-end ligatethe fragments to an appropriate site in coat polypeptide. In aparticular embodiment, a restriction site may be inserted into theAB-loop or N-terminus of the coat polypeptide). Only a minority ofclones will carry productive inserts, because they shift reading frame,introduce a stop codon, or receive an insert in antisense orientation,Any expression vector may in one embodiment contain a marker topre-select clones with intact coat coding sequences. For example,GalE-strains of E. coli are defective for galactose kinase andaccumulate a toxic metabolite when β-galactosidase is expressed in thepresence of the galactose analogue, phenyl-β,D-galactoside (PGaI).Subjecting a random antigen-fragment library to selection fortranslational repressor function in the GalE-strain CSH41 F-containingpRZ5, a plasmid that fuses the MS2 replicase cistron's translationaloperator to lacZ will eliminate most undesired insertions by enrichingthe library for those that at least maintain the coat reading-frame.

Synthesis

In a particular embodiment, the populations of the present invention maybe synthesized in a coupled in vitro transcription/translation systemusing procedures known in the art (see, for example, U.S. Pat. No.7,008,651 Kramer et al., 1999, Cell-free coupledtranscription-translation systems from E. coli, In. Protein Expression.A Practical Approach, Higgins and Hames (eds.), Oxford UniversityPress). In a particular embodiment, bacteriophage T7 (or a related) RNApolymerase is used to direct the high-level transcription of genescloned under control of a T7 promoter in systems optimized toefficiently translate the large amounts of RNA thus produced [forexamples, see Kim et al., 1996, Eur J Biochem 239: 88 1-886; Jewett etal., 2004, Biotech and Bioeng 86: 19-26].

It is possible in a mixture of templates, particularly in the populationof the present invention, different individual coat polypeptides,distinguished by their fusion to different peptides, could presumablypackage each other's mRNAs, thus destroying the genotype/phenotypelinkage needed for effective phage display. Moreover, because eachcapsid is assembled from multiple subunits, formation of hybrid capsidsmay occur. Thus, in one preferred embodiment, when preparing thepopulations or libraries of the present invention, one or more cycles ofthe transcription/translation reactions be performed in water/oilemulsions [Tawfik et al., 1998, Nat Biotechnol 16: 652-6]. In this nowwell-established method, individual templates are segregated into theaqueous compartments of a water/oil emulsion. Under appropriateconditions huge numbers of aqueous microdroplets can be formed, eachcontaining on average a single DNA template molecule and the machineryof transcription/translation. Because they are surrounded by oil, thesecompartments do not communicate with one another. The coat polypeptidessynthesized in such droplets should associate specifically with the samemRNAs which encode them, and ought to assemble into capsids displayingonly one peptide. After synthesis, the emulsion can be broken and thecapsids recovered and subjected to selection. In one particularembodiment, all of the transcription/translation reactions are performedin the water/oil emulsion. In another embodiment, mixed capsids may beobtained in one or more cycles of transcription/translation reactionsbut subsequent cycles of the transcription/translation reaction,particularly beginning with the second, third, fourth or fifth cycle,are carried out in the water/oil emulsion.

Uses of VLPs and VLP Populations

There are a number of possible uses for the VLPs and VLP populations ofthe present invention. As will be described in further detail below, theVLPs may be used to as immunogenic compositions, particularly vaccines,drug delivery devices, biomedical imaging agents and self-assemblingnanodevices. The VLP populations of the present invention may be used toselect suitable vaccine candidates.

Selection of Vaccine Candidates

The VLP populations or libraries of the present invention may be used toselect vaccine candidates. The libraries may be random or antigeniclibraries. A particular embodiment is outlined in FIG. 5. Libraries ofrandom or alternatively antigen-derived peptide sequences are displayedon the surface of VLPs, and specific target epitopes, or perhapsmimitopes are then isolated by affinity-selection using antibodies.Since the VLPs encapsidate their own mRNAs, sequences encoding them (andtheir guest peptides) can be recovered by reverse transcription and PCR.Individual affinity-selected VLPs are subsequently cloned,over-expressed and purified.

Techniques for affinity selection in phage display are well developedand are directly applicable to the VLP display system of the presentinvention. Briefly, an antibody (or antiserum) is allowed to formcomplexes with the peptides on VLPs in a random sequence or antigenfragment display library. Typically the antibodies will have beenlabeled with biotin so that the complexes can be captured by binding toa streptavidin-coated surface, magnetic beads, or other suitableimmobilizing medium. After washing, bound VLPs are eluted, and RNAs areextracted from the affinity-selected population and subjected to reversetranscription and PCR to recover the coat-encoding sequences, which arethen recloned and subjected to further rounds of expression and affinityselection until the best-binding variants are obtained. A number ofschemes for retrieval of RNA from VLPs are readily imagined. Oneattractive possibility is to simply capture biotin-mAb-VLP complexes instreptavidin coated PCR tubes, then thermally denature the VLPs andsubject their RNA contents directly to RT-PCR. Many obvious alternativesexist and adjustments may be required depending on considerations suchas the binding capacities of the various immobilizing media. Once theselected sequences are recovered by RT-PCR it is a simple matter toclone and reintroduce them into E coli, taking care at each stage topreserve the requisite library diversity, which, of course, diminisheswith each round of selection. When selection is complete, each clone canbe over-expressed to produce a VLP vaccine candidate.

Immunogenic Compositions

As noted above, the VLPs identified by the screening procedures of thepresent invention may be used to formulate immunogenic compositions,particularly vaccines. The vaccines should be in a form that is capableof being administered to an animal. Typically, the vaccine comprises aconventional saline or buffered aqueous solution medium in which thecomposition of the present invention is suspended or dissolved. In thisform, the composition of the present invention can be used convenientlyto prevent, ameliorate, or otherwise treat a condition or disorder. Uponintroduction into a host, the vaccine is able to provoke an immuneresponse including, but not limited to, the production of antibodiesand/or cytokines and/or the activation of cytotoxic T cells, antigen.presenting cells, helper T cells, dendritic cells and/or other cellularresponses.

Optionally, the vaccine of the present invention additionally includesan adjuvant which can be present in either a minor or major proportionrelative to the compound of the present invention. The term “adjuvant”as used herein refers to non-specific stimulators of the immune responseor substances that allow generation of a depot in the host which whencombined with the vaccine of the present invention provide for an evenmore enhanced immune response. A variety of adjuvants can be used.Examples include complete and incomplete Freund's adjuvant, aluminumhydroxide and modified muramyl dipeptide.

Optionally, the vaccine of the present invention additionally includesan adjuvant which can be present in either a minor or major proportionrelative to the compound of the present invention.

Targeted Drug Delivery

The MS2 VLP is a hollow sphere with an internal diameter on the order of20 nm. In a particular embodiment, the VLP comprises the drug, e.g., aprotein toxin to be delivered and optionally a ligand that binds tocell-type specific receptors. The internal composition of such aparticle may be controlled by specifically loading it, for example, witha protein toxin like ricin, by coupling it to a synthetic translationaloperator mimic. By conferring the ability to bind cell type-specificreceptors to the outer surface of such particles, it is possible totarget delivery of the toxin (or other drug) to selected cell types.

Biomedical Imaging Agents

In the same way that drugs can be targeted to specific cell types, socould contrast agents for magnetic resonance imaging be delivered tospecific cells or tissues, potentially increasing enormously thediagnostic power of MRI. In fact, MS2 particles have already beenlabeled with gadolinium to greatly increase MRI contrast [Anderson etal., 2006, Nano Letters 6(6), 1160-1164]. Thus, in a particularembodiment, such particles could be targeted to specific sites bydisplaying appropriate receptor-specific peptides on their surfaces.

Self-Assembling Nano-Devices

The VLPs of the present invention may comprise peptides with affinityfor either terminus of a filamentous phage particle that display metalbinding proteins. A VLP with affinity for either terminus of afilamentous phage particle would create the possibility of connectingthese spheres (and whatever they contain) to the ends of filamentousphage nanowires. Alternatively, the VLPs may display metal-bindingpeptides (e.g. gold and zinc) so that arrays with unusual electrical andoptical properties may be obtained. Alternatively, VLPs with improvedability to self-assemble into these arrays may be produced by displayingpeptides with affinity for a particular surface, or that alter theself-association properties of the VLPs themselves.

EXAMPLES

The invention may be better understood by reference to the followingnon-limiting examples, which are provided as exemplary of the invention.The following examples are presented in order to more fully illustratethe preferred embodiments of the invention and should in no way beconstrued, however, as limiting the broad scope of the invention.

Example 1 In vitro Expression of MS2 Coat Protein

Extracts of E coli cells capable of carrying out transcription andtranslation in vitro were described more than 30 years ago [Zubay G.,1973, Annu Rev Genet. 7: 267-87], but recent improvements have greatlyincreased their ability to produce useful quantities of product (seeKrarner et al., Cell-free coupled transcription-translation systems fromE. coli, In.: Protein Expression. A Practical Approach, Higgins andHames (eds.), Oxford University Press (1999) for example). Originally,these systems relied on the presence of endogenous E. coli RNApolymerase to transcribe genes from relatively weak promoters, and itwas necessary to utilize the incorporation of radioactive amino acidseven to detect the relatively low-level of protein they typicallysynthesized. Modern systems use bacteriophage T7 (or a related) RNApolymerase to direct the high-level transcription of genes cloned undercontrol of a T7 promoter in systems optimized to efficiently translatethe large amounts of RNA thus produced [for examples, see Kim et al.,1996, Eur J Biochem 239: 88 1-886; Jewett et al., 2004, Biotech andBioeng 86: 19-26]. An example result is shown in FIG. 6. Here an invitro transcription/translation system (in this case the Activeprosystem of Ambion, Inc.) was programmed with plasmids expressing MS2 coatprotein from a T7 promoter, and the proteins were separated bySDS-polyacrylamide gel electrophoresis. Lane 3 shows the plasmid calledpETCT (FIG. 20) directed the synthesis of coat protein in sufficientquantity that it was easily visualized in the stained gel. Probing aWestern blot of a similar gel with anti-MS2 serum confirmed theproduct's identity (FIG. 6). In lane 4 is shown the protein synthesizedusing pETMCT, a plasmid differing from pETCT by the inclusion of thetranslational operator downstream of the coat coding sequence. Itappears that pETMCT synthesizes significantly less coat protein thanpETCT. Still, the product is easily detected. Electrophoresis of thetranscription/translation products in an agarose gel, where correctlyassembled viruses and virus-like particles have a distinctive mobility,reveals material having the mobility and immunological reactivitycharacteristic of MS2 capsids (FIG. 7). Although much of the coatprotein seems to remain unassembled, significant quantities of capsidsare present in both samples. Furthermore, the appearance of an ethidiumbromide stained capsid band indicates that the particles package RNA.

Example 2 Development of MS2 VLP Display A-B Loop Insertion

The AB-loop is a 3-residue turn connecting coat protein's A and Bbeta-strands (FIG. 8). Peptides inserted here are highly accessible and,because they are tethered at both ends, conformationally constrained.Since many epitopes in their native environments are found in surfaceloops, this is a natural location for peptide display. Capsid geometrydictates that the AB-loop is encountered at regular intervals of roughly30 angstroms, so peptides inserted here form dense repetitive arrays.Efforts to produce active AB-loop insertions have met with mixed success[Stockley et al., 2000, Methods Enzymol 326:551-569]. In some cases,insertions were tolerated, but when they were not, the protein failed tofold correctly and either aggregated in inclusion bodies or wasproteolyically degraded. In the instant example, a solution isdescribed.

Two examples are presented to illustrate the point. First, to facilitateconstruction of insertions introduced two silent mutations wereintroduced in coat protein codons 14 and 15 to produce a convenient andunique Kpn I site within the AB-loop-encoding sequence. Two different10-amino acid peptides were inserted here. The first, called ECL2, isderived from extracellular loop-2 of the HIV co-receptor, CCR5. Theother (V3) is from the third variable loop of the HIV envelopeglycoprotein, gp120. Both insertions interfered with coat proteinfolding. In fact, the proteins failed even to accumulate in significantquantities apparently because they were degraded. These disappointingresults would argue against the use of MS2 for epitope display were itnot for a simple trick that reverses these folding/stability defects.Inspection of the 3-dimensional structure of the coat protein dimer(FIG. 9) reveals the close physical proximity of the N-terminus of onechain to the C-terminus of the other. These ends are covalently linkedby duplicating the coat sequence and fusing the two copies into one longreading frame, thus producing “dimers” whose two halves are synthesizedas a single polypeptide chain. This so-called single-chain dimer has allthe functions of normal coat protein; it folds correctly, repressestranslation by specifically binding the translational operator of thereplicase cistron, and assembles into a normal VLP. It was originallyconstructed to analyze the RNA binding site of mutant coat proteins inheterodimer complementation experiments [Powell et al., 2001, BMC MolBiol 2:6; Peabody et al., 1999, J Biol Chem 274(36):25403-25410; Peabodyet al., 1996, Nucleic Acids Res 24(12):2352-2359], but while performingthat work it was noticed that the single-chain dimer is substantiallymore stable thermodynamically than its parent. It is considerably moreresistant to thermal and chemical denaturation and is dramatically moretolerant of various mutational perturbations [for examples, see Peabodyet al., 1999, J Biol Chem 274(36):25403-25410] and Peabody et al., 1997,Arch Biochem Biophys 347(1): 85-921 Although inserting the ECL2 or V3epitope into the AB-loop of wild-type coat protein disrupts its abilityto properly fold, both peptides are tolerated when inserted into oneAB-loop of a single chain dimer, They are produced as soluble proteinsin normal yields, they fold correctly, and they assemble into VLPs withthe foreign epitopes on their outer surfaces.

Although the density of displayed epitopes is reduced by half whenpresented in only one AB-loop of the single-chain dimer these particlesretain their high immunogenicity. Immunization of mice with MS2-V3-VLPs,even in the absence of exogenous adjuvant, induced high titer antibodiesable to recognize the V3 peptide (FIG. 12). Further, the anti-V3 seraprotected cells against HIV infection in vitro, The MS2-ECL2-VLPselicited a similarly strong immune response to CCR5. These resultssuggested that the single-chain dimer might provide the means to producepotently immunogenic MS2 VLPs tolerant of a wide range of peptideinsertions in the AB-loop.

In addition to those described above, several other designed peptideshave been inserted into one AB-loop of a single chain MS2 coat proteindimmer, and in nearly every case translational repression and capsidassembly activities remained intact, suggesting a broad tolerance of thesingle-chain dimmer to such insertions.

N-Terminal Fusions

The N-terminus presents an alternative site of peptide fusion. FIGS. 8and 9 show the locations of the N-termini and demonstrates theiraccessibility at the VLP surface. Three different specific fusions wereproduced, including an 8-amino acid flag epitope, a 20-amino acid6×His-tag with a thrombin cleavage sequence, and a 23-amino acid biotinligase target peptide. In each case, the protein remains functional fortranslational repression, indicating that it folds correctly into thedimeric structure necessary for specific RNA binding. However, thepresence of the fusion tends to interfere with assembly of dimers intoVLPs, presumably because the elongated N-termini of three differentsubunits crowd each other at quasi-3-fold symmetry axes in the capsid.But again the single-chain dimer corrects the defect, because reducingby half the number of N-termini apparently diminishes crowding andpermits assembly. The VLPs thus produced display the foreign peptide ontheir outer surfaces [Peabody, 1997, Arch Biochem Biophys 347(1):85-92].Functional coat protein-GFP fusions have also been produced for variouspurposes, indicating that fusion even of large proteins permits coatprotein folding. Thus, the N-terminus provides a second site suitabletarget for peptide display.

The N-terminus provides a means to display conformationallyunconstrained peptides, which, because they are free to adopt a widerrange of conformations, may increase the likelyhood that a randomsequence library contains a peptide capable of recognizing anyparticular antibody.

Example 3 Immunogenic Display of Diverse Peptides on Virus-LikeParticles of RNA Bacteriophage MS2

In the instant example, a platform is described for vaccine developmentbased on the VLPs of RNA bacteriophage MS2. It serves for the engineereddisplay of specific peptide sequences, but also allows the constructionof random peptide libraries from which specific binding activities canbe recovered by affinity selection. Peptides representing the V3 loop ofHIV gp120 and the ECL2 loop of the HIV coreceptor, CCR5, were insertedinto a surface loop of MS2 coat protein. Both insertions disrupted coatprotein folding and VLP assembly, but these defects were efficientlysuppressed by genetically fusing coat protein's two identicalpolypeptides into a single-chain dimer. The resulting VLPs displayed theV3 and ECL2 peptides on their surfaces where they showed the potentimmunogenicity that is the hallmark of VLP-displayed antigens.Experiments with random-sequence peptide libraries show the single-chaindimer to be highly tolerant of 6-, 8- and 10-amino acid insertions. Notonly do MS2 VLPs support the display of a wide diversity of peptides ina highly immunogenic format, but they also encapsidate the mRNAs thatdirect their synthesis, thus establishing the genotype/phenotype linkagenecessary for recovery of affinity selected sequences. The single-chainMS2 VLP therefore unites in a single structural platform the selectivepower of phage display with the high immunogenicity of VLPs.

Materials and Methods Plasmid Construction

A PCR overlap extension method [Higuchi et al., 1988, Nucleic Acids Res16(15):7351-67] introduced two silent nucleotide changes in codons 14and 15 of the coat sequence and a unique KpnI site into the MS2 coatgene of pMCT, a plasmid nearly identical to the previously describedpCT119 [Peabody, 1990, J Biol Chem, 265(10):5684-9]. The new constructis called pMCTK2 (see FIG. 18). Synthetic duplex oligonucleotides (fromIntegrated DNA Technologies, see FIG. 10B) encoding the ECL2 and V3peptides were inserted into the Kpn I site (SEQ ID NOS:4-9). DNAsequence analysis of the resulting recombinants confirmed the presenceof the designed sequences. These plasmids were called pMCTK-ECL2 andpMCTK-V3 (FIG. 10A).

The various single-chain dimer versions of the pMCTK-ECL2 and -V3recombinants (FIG. 10A) were produced by duplication of the wild-typeand recombinant coat sequences. Briefly, the upstream half was producedby PCR amplification using a 5′ primer that anneals to plasmid sequencesupstream of the coat sequence, and a downstream primer that creates aBgl I site at the 3′-end of the coding sequence. This fragment wasdigested at the new Bgl I site and at the Hind III site in the upstreamplasmid sequence. The downstream half was synthesized using a primerthat creates a Bgl I site at the 5′-end of the coding sequence, and a3′-primer that anneals to plasmid sequences downstream of coat. Thisfragment was digested at the Bgl I site at the 5′-end of this PCRfragment and at a Bam HI site present in plasmid sequences downstream ofcoat. These two DNAs were then joined by ligation to a vector fragmentderived by Hind III-Bam HI cleavage of pMCT and introduced into E. coliby transformation. The resulting plasmids contain a duplication of thecoat sequence, with the C-terminal amino acid of the upstream copy fusedto amino acid 2 of the downstream sequence. This arrangement isidentical to that found in the previously constructed p2CT-dl13 [Peabodyet al., 1996, Nucleic Acids Res 24(12):2352-9], but with twoconservative amino acid substitutions to accommodate the introduction ofthe Bgl I site, whose presence at the junction simplifies single-chaindimer construction. The plasmid p2MCTK3 was constructed by a similarprocess. It provides a single-chain dimer with a unique Kpn I site inthe AB-loop of its downstream half.

Protein Expression, Purification and Functional Assays

To test the recombinant proteins for translational repressor activity,each plasmid was introduced into E. coli strain CSH41F⁻ containing thetranslational repression reporter plasmid called pRZ5 [Peabody, 1990, JBiol Chem, 265(10):5684-9] and plated on LB medium containing theβ-galactosidase chromogenic substrate,5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal). To determine theexpression levels of recombinant proteins and their solubilities, celllysates from 1 ml overnight cultures were separated into soluble andinsoluble fractions and subjected to SDS-gel electrophoresis (seePeabody, 1997, Arch. Biochem. Biophys. 347(1):85-92 for details).Contents of the gel were transferred to a nitrocellulose membrane andprobed with rabbit anti-MS2 serum and alkaline phosphatase-conjugatedgoat anti-rabbit IgG antibodies. The coat proteins encoded in p2MCTK3(see FIG. 19) p2MCTK-ECL2 and p2MCTK-V3 were purified to greater than90% purity by chromatography in Sepharose CL-4B using methods describedpreviously [Peabody, 1990, J Biol Chem, 265(10):5684-9].

Rapid assessment of a recombinant protein's ability to assemble into aVLP is performed by electrophoresis of sonicated cell lysates (from 1 mlovernight cultures) in gels of 1% agarose in 50 mM potassium phosphate,pH 7.5 [Peabody, 1993, Embo J 12(2):595-600]. Gels are stained withethidium bromide to reveal the presence of VLPs, which contain hostRNAs. The identity of the VLPs is then confirmed by transferring thecontents of the gel to nitrocellulose and probing with rabbit anti-MS2serum and an alkaline phosphatase-labeled second antibody.

Libraries of Random Sequence Peptides

To insert random DNA sequences encoding 6, 8 and 10-amino acid peptidesinto the AB-loop, the primers described below were used to amplify acoat fragment from pMCT in three different PCR reactions. Threedifferent 5′-primers [called (NNY)₆, (NNY)₈ and (NNY)₁₀] attach at codon14 a Kpn I site and 6, 8 or 10 randomized codons of sequence NNY (whereN=A,C,G, or T and Y=T or C). Each reaction employed a single 3′-primerthat annealed downstream of a Bam HI site in the plasmid vector. Theresulting PCR products were digested with Kpn I and Bam HI, gel purifiedand ligated to the similarly digested vector fragments of p2MCTK3 (seeFIG. 19) or pMCTK2 (see FIG. 18). These were introduced bytransformation into strain CSH41F− containing plasmid pRZ5 [Peabody,1990, J Biol Chem 265(10):5684-9] and plated on LB medium containingX-gal. Control ligations containing only vector DNA gave rise to atleast 1000-fold fewer colonies than those that contained an insertfragment. After overnight incubation at 37° C. the relative numbers ofblue and white colonies obtained were determined by counting. Properlyfolded coat proteins repress translation of β-galactosidase and yieldwhite colonies. From each library 24 white and 12 blue colonies werepicked to two different 1 ml cultures in LB medium and grown overnightwith shaking at 37° C. One set of cultures was used for plasmidisolation and DNA sequence analysis. The other was lysed by sonicationand subjected to agarose gel electrophoresis as described above. VLPswere visualized by ethidium bromide staining and by blotting tonitrocellulose and probing with rabbit anti-MS2 serum and alkalinephosphatase-conjugated goat anti-rabbit IgG.

Packaging of Coat-Specific RNAs

The Xba I-Bam HI fragments of plasmids pCT119 [Peabody, 1993, Embo J12(2):595-600] and p2CTdl-13 [Peabody et al., 1996, Nucleic Acids Res24(12):2352-9] were inserted into the T7 expression vector, pET3d[Studier et al., 1990, Methods in Enzymology 185:60-89]. Coat proteinexpression was induced by IPTG in bacterial strain BL21(DE3)/pLysS usingstandard methods, and VLPs were extracted and purified by SepharoseCL-4B chromatography [Peabody, 1990, J Biol Chem 265(10):5684-9]followed by centrifugation to equilibrium in CsCl gradients (1.40 g/ccstarting density) at 40,000 rpm in the SW50.1 rotor. RNAs were extractedfrom VLPs using phenol/chloroform and applied to a 1.5% agarose gelcontaining formaldehyde [Lehrach et al., 1977, Biochemistry16:4743-4751]. The geL was blotted to nitrocellulose and probed with acoat-specific synthetic oligonucleotide(5′-CGAGTTAGAGCTGATCCATTCAGCGACCCC-3′) (SEQ ID NO:10) labeled at its5′-end with ³²P. Control RNAs were produced by transcription of pETCT(see FIG. 20) and pET2CTdl-13 (see FIG. 21-How does pET2CTdl-13 differfrom pET2CT?) in vitro using T7 RNA polymerase.

Immunization and Characterization of Antisera

Antisera were prepared by inoculating C57Bl/6 mice with 15 μg wild-typeMS2 VLPs, 15 μg MS2-V3 VLPs, or 15 μg MS2-ECL2 VLPs. Mice wereinoculated intramuscularly three times at 2-week intervals. Sera werecollected prior to each injection and 2 weeks after the final boost.When adjuvant was used, antigen was diluted 1:1 in complete Freund'sadjuvant (CFA; initial injection) or incomplete Freund's adjuvant (IFA;subsequent boosts) immediately prior to the injection. All animal carewas in accordance with the National Institutes of Health and Universityof New Mexico guidelines. Antibody titers were determined by ELISA usingpeptides corresponding to the target sequences. A V3 peptide(RIQRGPGRAFVTGK (SEQ ID NO:11); synthesized by CommonwealthBiotechnologies, Chantilly, Va.) was conjugated to KLH using acarbodiimide crosslinker (Pierce). A cyclic peptide corresponding tomacaque CCR5 ECL2 (C₁D₂R₃S₄Q₅R₆E₇G₈L₉H₁₀Y₁₁T₁₂G₁₃, in which Gly13 waslinked to Asp2 through a dipeptide spacer; synthesized by CeltekPeptides, Nashville Tenn.) was conjugated to avidin using aheterobifunctional crosslinker (SMPH; Pierce). Conjugated peptides wereimmobilized (at 200 ng/well) onto Immulon II ELISA plates (DynexTechnologies, Chantilly, Va.) overnight at 4° C. and then wells wereblocked with PBS plus 0.5% non-fat dry milk for 2 h at room temperature.Mouse serum was serially diluted in PBS-0.5% milk and applied to wellsfor 2.5 h at room temperature. Reactivity to antigen was determinedusing horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (JacksonImmunoresearch, West Grove, Pa.) at a 1:2000 dilution in blocking bufferas a secondary antibody. Upon development, optical densities were readat 405 nm using an OpSys MR plate reader (Thermo Labsystems, Waltham,Mass.). OD₄₀₅ values that were greater than twice background(usually >0.080) were considered positive.

HIV neutralization was measured using the MAGI-CCR5 indicator cell line.These cells and the MAGI-CCR5 assay are described in more detail byChackerian et al., 1997, J Virol 71(5):3932-3939. One hour prior toinfection, dilutions of mouse sera were incubated with approximately 100infectious HIV-1_(LAI) virus particles in a total volume of 50 μL at 37°C. The virus-antibody mixture was then added to wells in a total volumeof 200 μL in the presence of 10 μg/mL DEAE-Dextran (Sigma-Aldrich, St.Louis, Mo.). After 2 h at 37° C., virus and antibody were removed fromeach well and replaced with 0.5 mL of media. Two days after infection,cells were fixed, washed, and stained for β-galactosidase activity, asdescribed previously [Kimpton et al., 1992, J Virol 66(4):2232-2239].

Binding of mouse serum IgG to native CCR5 was tested by flow cytometry.293T cells were transiently transfected with a rhesus macaqueCCR5-encoding expression vector [pc.Rh-CCR5; [Chen et al., 1997, J Virol71(4):2705-14]]. Cells were detached from the monolayer using 5 mM EDTAand then washed three times in staining buffer (PBS plus 0.5% BSA). Toremove antibodies that bound non-specifically to cells, sera waspreincubated with untransfected 293T cells (10⁵ cells for every 5 μL ofsera) for 45 min at 4° C. Sera was removed from cells and then incubatedwith CCR5- or mock-transfected cells. Approximately 10⁵ cells wereresuspended in 50 μl of staining buffer plus 10 μL of mouse sera for 30min at 4° C. After washing three times with staining buffer, cells wereresuspended in 50 μl of staining buffer plus 250 ng of fluoresceinisothiocyanate (FITC)-labeled goat anti-mouse IgG (JacksonImmunoresearch) and then incubated for 30 min at 4° C. As a control,cells were stained with secondary antibody alone or with a phycoerythrin(PE)-labeled anti-CCR5 monoclonal antibody (3A9; BD Pharmingen). Beforeanalysis, cells were washed twice more with staining buffer andresuspended in 0.5 ml of staining buffer. Specific binding was measuredrelative to mock-transfected cells.

Results Insertion of the ECL2 and V3 Peptides in the Coat ProteinAB-loop

The surface accessibility and regular geometric spacing of the AB-loopin the MS2 VLP make it an attractive site for the display of foreignpeptides (FIG. 8). Two model peptides that in their natural environmentsare found in exposed loops have been inserted. One was derived from theV3 loop of the HIV envelope protein, gp120 of the lab-adapted strain,HIV-1_(LAI). Its core sequence is relatively conserved among HIVisolates, and is a target of neutralizing antibodies [Laman et al.,1992, J Virol 66(3):1823-31]. The other peptide comes from the secondextracellular loop (ECL2) of the macaque chemokine receptor, CCR5. Inaddition to its role in immune chemotaxis, CCR5 is a major coreceptorused by HIV to enter target cells. This particular sequence represents aregion of ECL2 referred to as the undecapeptidyl arch (UPA) and isinvolved in HIV entry into cells [Misumi et al., 2001, J Virol75(23):11614-20].

To facilitate peptide insertion, the plasmid pMCTK2 was constructed (seeFIG. 18). It is similar to the previously described pCT119 [Peabody,1990, J Biol Chem 265(10):5684-9], but, following the example of Masticoet al. [Mastico et al., 1993, J Gen Virol 74 (Pt 4):541-8], wasengineered to contain a Kpn I site in the AB-loop-encoding sequence(FIG. 10A). We then inserted duplex oligonucleotides (FIG. 10B) encoding10-amino acid V3 and ECL2 peptides. Because insertion at Kpn I resultsin duplication of codons 14 and 15, the length of coat protein wasactually increased by a total of twelve amino acids. The resultingplasmids, pMCTK2-ECL2 and pMCTK2-V3 express the recombinant coatproteins from the E. coli lac promoter. A Western Blot of the solubleand insoluble fractions of crude cell lysates (FIG. 11A) shows that thewild-type protein was abundantly produced in a predominantly solubleform, but neither of the recombinant proteins was present in detectablequantities. Their absence may be due to proteolytic degradation as asecondary consequence of a severe folding defect.

Functional tests confirm that the V3 and ECL2 recombinants aredefective. Coat protein normally serves as a translational repressor,shutting off synthesis of the viral replicase by binding an20-nucleotide RNA hairpin containing its ribosome binding site (theso-called translational operator). Fusing this sequence to the E. colilacZ gene on the plasmid called pRZ5 [Peabody, 1990, J Biol Chem265(10):5684-9] provides a simple means of assessing translationalrepressor activity of coat protein variants. Cells containing pRZ5 formwhite colonies on x-gal plates when they express functional coatprotein, but make blue colonies they do not. Neither the ECL2- orV3-containing recombinant proteins inhibited β-galactosidase synthesisat all, indicating a complete failure to repress translation (Table I).

TABLE 1 Blueness on XGal pUCter3 +++ pCT119 − p2MCTK3 − pCT-ECL2 +++pCT-V3 +++ p2M-ECL2-2 +++ p2M-V3-2 +++ p2M-ECL2-1 − p2M-V3-1 −

The Folding Defects are Corrected in Single-Chain Dimers

FIGS. 8 and 9 show the structure of the coat protein dimer andillustrates the structural basis for construction of single-chaindimers. Note the physical proximity of the C-terminus of one subunit tothe N-terminus of the other. It was previously demonstrated that geneticfusion of the two chains into single-chain dimers greatly protects theprotein against the destabilizing effects of amino acid substitutionsand chemical denaturants [Peabody, 1997, Arch Biochem Biophys347(1):85-92, Mastico et al., 1993, J Gen Virol 74 (Pt 4):541-8, Peabodyet al., 1996, Nucleic Acids Res 24(12):2352-9]. In an effort to suppressthe defects imparted by the ECL2 and V3 insertions, we constructed twotypes of single-chain dimer; one has a foreign peptide in both AB-loops,and the other contains the peptide in only its C-terminal half (FIG.10A).

The expression of the single-chain proteins was assessed by Western Blot(FIG. 11A). When present in both AB-loops, the single-chain dimer seemsto revert partially the defects caused by the foreign peptides in theconventional dimer. The recombinant proteins are now detectable, butthey are found predominantly as insoluble aggregates, suggesting thatthey are mostly misfolded. Their failure to correctly fold is alsosuggested by an inability to repress translation (Table 1). However,when the foreign peptides are incorporated into only the downstream copyof the single-chain dimer's two AB-loops the defect is fully corrected.The proteins are produced in normal amounts, are found mostly in thesoluble fraction of the cell, and they repress translation just likewild-type (Table 1). The elution of the ECL2 and V3 single-chainproteins from Sepharose CL-4B at the same position as authentic MS2virus is another indication that the recombinant proteins assemblenormally into VLPs (FIG. 11B), and also provided a means to purify theECL2 and V3 recombinant VLPs [Peabody, 1990, J Biol Chem265(10):5684-9]. Analysis by SDS-polyacrylamide gel electrophoresisshows that the VLPs are nearly free of contaminating cellular proteins.Electrophoresis of the VLPs in an agarose gel under native conditions isshown in FIG. 11C. As expected of a properly assembled VLP, eachcontains RNA (it stains with ethidium bromide) and exhibits an alteredelectrophoretic mobility due to the charge differences conferred by theECL2 and V3 peptides (FIG. 11B). Staining of the gel with the proteinstain Coomassie Brilliant Blue shows the same pattern. The mobility ofthe capsid produced by the unmodified single-chain dimer (p2MCTK3-seeFIG. 19) is slightly greater than that shown by the wild-type VLPproduced from pMCTK, perhaps because subunit fusion reduces by half thenumber of positively charged N-termini, which happen to reside near theVLP surface.

Recombinant V3-VLPs were further characterized using a monoclonalantibody (mAb) (MAbIIIB-V3-13) that recognizes the V3 epitope and hasHIV neutralizing activity [Laman et al., 1992, J Virol 66(3):1823-31].MAbIIIB-V3-13 specifically bound to purified V3-VLPs immobilized on aELISA plate, but not to wild-type MS2 VLPs or ECL2-VLPs (FIG. 12A),showing that the inserted V3 sequence is exposed on the surface ofrecombinant VLPs in a form competent for recognition by the monoclonalantibody.

Immunogenicity of the ECL2 and V3 Recombinants

The abilities of purified recombinant ECL2- and V3-VLPs to induceantibodies against the target sequences were assessed by immunization ofC57Bl/6 mice. Sera were tested for IgG antibodies specific for eitherthe V3 or ECL2 peptides by end-point dilution ELISA. Mice immunized withV3-VLPs or ECL2-VLPs developed high titer (>10⁴) IgG responses againstthe corresponding peptide, but not against the heterologous peptide(FIG. 12B). No peptide-reactive antibodies were detected in sera fromwild-type MS2 VLP-immunized mice. As with other VLP-based immunogens,high titer antibodies were induced without the use of exogenousadjuvants; coadministration of FA boosted IgG levels only slightly.

It was next determined whether induced anti-V3 antibodies bound tofull-length native protein. Because monoclonal antibodies that bind tothis region of V3 have HIV neutralizing activity [Misumi et al., 2001, JVirol 75(23):11614-20], we tested whether sera from V3-VLP immunizedmice could inhibit HIV infection. Pooled sera from V3-VLP immunizedmice, control sera, or two different HIV neutralizing monoclonalantibodies were preincubated with approximately 100 infectiousHIV-1_(LAI) particles, which were then used to infect an HIV indicatorcell line (MAGI cells). Control sera from mice immunized with wild-typeMS2 VLPs had no HIV neutralizing activity whereas sera from V3-VLPimmunized mice neutralized HIV (˜75% neutralization at a 1:10 seradilution) (FIG. 12C). The more potent neutralizing activity displayed bythe anti-V3 mAb (MAbIIIB V3-13) is consistent with the ˜10-fold higherV3-peptide ELISA binding activity of this mAb relative to the V3-VLPsera.

The ability of ECL2-VLPs to elicit antibodies that bind native CCR5 wastested by flow cytometry. Macaque CCR5 was expressed on 293T cells bytransient transfection with a rhesus macaque CCR5 expression vector(pc.Rh.CCR5), and the binding of mouse IgG was measured relative tomock-transfected cells. As shown in FIG. 12D, sera from ECL2-VLPimmunized mice bound to CCR5-transfected cells (relative tomock-transfected cells) whereas sera from control mice did not,demonstrating that anti-ECL2 antibodies bind native CCR5.

Testing the Single-Chain Dimer's Tolerance of Random Peptide Insertions

A library of random 6-amino acid insertions in the AB-loop of pMCTK2(wild-type coat) and of 6-, 8-, and 10-amino acids in the second AB-loopof p2MCTK3 (the single-chain dimer) was created. This was accomplishedby insertion of 6, 8 or 10 copies of the sequence NNY, where N=anynucleotide, and Y=C or T. Random NNY triplets produce codons for 15 ofthe 20 amino acids, and although such libraries cannot encode lys, glu,gln, trp and met, they create considerable diversity while avoiding theintroduction of stop codons. The libraries were introduced into strainCSH41F−/pRZ5 and plated on X-Gal plates where the ability of coatprotein to repress translation of β-galactosidase resulted in theformation of white colonies. Failure to repress, on the other hand,gives blue colonies.

The results are shown in Table 2.

TABLE 2 Percent white colonies pMCTK2 p2MCTK3 (NNY)₆ 2 96 (NNY)₈ nd 94(NNY)₁₀ nd 92

These results dramatically illustrate the importance of subunit fusionfor successful folding. In the case of the pMCTK2 6-mer library only 2%of colonies were white, showing that only rarely was a 6-mer insertiontolerated in the AB-loop of the conventional dimer. On the other hand,in the single-chain dimer 96% of 6-mer, 94% of 8-mer, and 92% of 10-merinsertions gave functional translational repressors. From each of theselibraries 12 blue recombinants were randomly selected for sequenceanalysis. It was found in the 6-mer and 8-mer libraries, that about halfthe defective clones (5/12 and 6/12, respectively) had frameshiftmutations (presumably caused by occasional errors during synthesis ofthe NNY primers), or were the products of anomolous ligation events. Onequarter (3/12) of the defective 10-mer clones also had frameshiftmutations. Thus, in each library a significant percentage ofrepressor-defective clones were not the results of failure to toleratepeptide insertions, but had other defects.

Visual inspection of the small percentage of sequences that resulted infolding failures immediately led to the impression that they areenriched in hydrophobic amino acids. This intuition was confirmed whenall the peptide sequences were joined together into a single 662-aminopolypeptide, with the tolerated (from white colonies, residues 1-482)and non-tolerated sequences (from blue colonies, residues 483-662)grouped together. A Kyte-Doolittle hydrophobicity plot [Gasteiger etal., 2005, The Proteomics Protocols Handbook, ed. J. M. Walker, pp.571-607; Kyte et al., 1982, Journal of Molecular Biology, 157:105-132]shows a distinct transition to higher average hydrophobicity at thewhite/blue junction (FIG. 13).

A number of white clones were picked from each of the 6-mer, 8-mer and10-mer libraries and subjected to analysis of their abilities to supportthe synthesis of properly assembled VLPs. Sonicated cell lysates weresubjected to agarose gel electrophoresis, and VLPs were visualized byethidium bromide staining (upper half of each set in FIG. 14) and by aWestern Blot probed stained with anti-MS2 serum and alkalinephosphatase-conjugated anti-rabbit IgG (lower half of each set. Themobilities of the recombinant VLPs are diverse, because, as sequenceanalysis shows, most of the inserted peptides contain at least onecharged amino acid. Importantly, tolerance of insertions is high: 100%(21/21) of 6-mer clones, and 87% (20/23) 8-mer clones contained adetectable VLP, while the frequency of successful 10-mers was 80%(16/20).

MS2 VLPs Encapsidate their mRNAs

Plasmids called pETCT and pET2CTdl-13 express wild-type coat protein andthe single-chain dimer, respectively, from transcripts of predictedlengths of about 580 and 970 nucleotides, whose ends are specified bythe T7 promoter and terminator sequences. VLPs were purified frombacteria, and their RNAs were extracted and subjected them to denaturingagarose gel electrophoresis [Lehrach et al., 1977, Biochemistry,16:4743-4751]. The ethidium-bromide-stained gel (FIG. 15, left panel)shows that each VLP contains a dominant species that comigrates with RNAmarkers produced by T7 transcription in vitro of the same pETCT andpET2CTdl-13 plasmids. Three other RNAs were also run as molecular weightmarkers and as hybridization controls. Two were genome RNAs extractedfrom purified MS2 (3,569 nucleotides) and Qβ phages (4,220 nucleotides),and another, about 650 nucleotides long, was produced by transcriptionin vitro of a plasmid containing HCV core sequences. A Northern Blotprobed with a ³²P-labeled synthetic coat-specific oligonucleotideverifies the identities of the encapsidated RNAs; only RNAs expected tocontain the coat sequence hybridize with the probe (FIG. 15, right).

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1-9. (canceled)
 10. An isolated transcription unit comprising abacterial or bacteriophage promoter, a coding sequence of an RNAbacteriophage single chain coat polypeptide dimer with a site forinsertion of a heterologous peptide in the downstream subunit of thedimer and a bacterial or bacteriophage terminator. 11-15. (canceled) 16.An isolated transcription unit comprising a bacteriophage promoter, acoding sequence of an RNA bacteriophage single chain coat polypeptidewith a site for insertion of a heterologous peptide in said codingsequence and optionally a bacteriophage terminator.
 17. A method forconstructing a library of virus-like particles, wherein each virus-likeparticle comprises a heterologous peptide in the upstream or downstreamsingle-chain RNA bacteriophage coat polypeptide dimer comprising (a)providing a plurality of transcription units of claim 10 (b) treatingsaid transcription units of (a) with a restriction enzyme; (c) insertingcoding sequences for heterologous peptides into said transcription unitsto obtain a population of transcription units (d) expressing saidtranscription units of (c) and (e) isolating said library.
 18. Themethod according to claim 17 wherein said transcription units areexpressed in a coupled transcription translation system in vitro. 19.The method according to claim 17 wherein said heterologous peptide is atleast four amino acid units in length.
 20. The method according to claim17, wherein said transcription units are expressed in a coupledtranscription translation system in vitro, wherein at least one cycle ofcoupled transcription/translation is conducted in a compartmentalizedwater/oil emulsion.
 21. A method for identifying a peptide having aproperty of interest comprising (a) providing a population of virus-likeparticles (VLPs), wherein each particle (a) is a VLP of an RNAbacteriophage, (b) comprises a single chain dimer coat polypeptide ofsaid phage modified by insertion of a heterologous peptide, wherein saidheterologous peptide is displayed on said VLP; (c) encapsidates saidphage mRNA, and wherein said coat polypeptide comprises an upstream anddownstream subunit; (b) assaying heterologous peptides expressed on theVLPs in said population for the property of interest to identify thepeptide having a property of interest.
 22. The method according to claim21, wherein the population in (a) is obtained by expressing atranscription unit comprising a bacteriophage promoter and 3′terminator, a coding sequence for a modified RNA bacteriophage coatpolypeptide, wherein said modification is a heterologous peptidesequence at least 4 amino acids in length in a coupledtranscription/translation system from a nucleic acid template in acompartmentalized water/oil emulsion and recovering said population fromsaid water/oil emulsion.
 23. The method according to claim 21, whereinsaid peptide having a property of interest is an immunogenic peptide.24. The method according to claim 21, wherein said peptide having aproperty of interest is an immunogenic fragment of a self-antigen. 25.The method according to claim 22, wherein said immunogenic fragment is afragment of an immunogenic HIV peptide.
 26. The method according toclaim 21, wherein said peptide having a property of interest is amimitope.
 27. A method for isolating an immunogenic protein comprising:(a) identifying said immunogenic peptide from a population of VLPsaccording to the method of claim 21; b) amplifying said identifiedimmunogenic peptide and (c) isolating said immunogenic peptide.
 28. Themethod according to claim 22, wherein said immunogenic peptide isisolated by affinity selection. 29-33. (canceled)
 34. An immunogeniccomposition comprising one or more VLPs of a MS-2 RNA bacteriophage andcomprises a single chain dimer of the coat polypeptide of said phage,said coat polypeptide comprising an upstream and downstream subunit,wherein said upstream or downstream subunit is modified by insertion ofan immunogenic heterologous peptide in either the upstream or downstreamsubunit of said dimer.
 35. The immunogenic composition of claim 34,wherein said heterologous peptide is a self-antigen or immunogenicfragment thereof.
 36. The immunogenic composition of claim 34, whereinsaid self-antigen is a peptide derived from the group consisting ofErbB-2, amyloid-beta, immunoglobulin E (IgE), gastrin, ghrelin, vascularendothelial growth factor (VEGF), interleukin (IL)-17, IL-23, IL-13,CCR5, CXCR4, nerve growth factor (NGF), angiotensin II, TRANCE/RANKL, orMUC-1.
 37. The immunogenic composition of claim 34, wherein saidheterologous peptide is an HIV immunogenic peptide.
 38. The immunogeniccomposition of claim 34, wherein said heterologous peptide is insertedin the downstream subunit of said coat polypeptide.
 39. The immunogeniccomposition of claim 34, wherein said heterologous peptide is insertedinto the AB-loop of said downstream subunit of said coat-polypeptide.40. The immunogenic composition of claim 34 wherein said heterologouspeptide is inserted into the N-terminus of said dimer of said coatpolypeptide.
 41. The method according to claim 17 wherein saidvirus-like particle comprises a heterologous peptide in the downstreamsingle-chain RNA bacteriophage coat polypeptide dimer.
 42. The methodaccording to claim 28 wherein said identified peptide is amplified byreverse transcription and polymerase chain reaction of RNA isolated fromthe affinity selected VLPs.